Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function

Objective To test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (osmotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage. Design Ejaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 800°C/min to −80°C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400°C/min (experiment 1) or either 1°C or 400°C/min (experiment 2). After warming, sperm were assessed for survival using motility as the endpoint in experiment 1 and motility, plasma membrane integrity, and mitochondrial function in experiment 2. Results In experiment 1, over the various cooling rates with a standard 400°C/min warming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10°C/min cooling rate. In experiment 2, over the various cooling rates, both 1 and 400°C/min warming rates produced similar but shifted plots of motility, plasma membrane integrity, and mitochondrial function versus cooling rate, which also produced inverted U-shaped patterns (n = 11). Maximal survival for each of the three endpoints occurred at 10°C/min cooling rate for the rapidly warmed sperm and at 1°C/min for the slowly warmed sperm. Conclusions These data support the hypotheses that a two-factor hypothesis of cryodamage applies to human spermatozoa and that an interaction exists between cooling rate and warming rate. These data also suggest that motility, plasma membrane integrity, and mitochondrial function are not differently affected by cooling and warming during cryopreservation.